1. Field of the Invention (Technical Field)
This invention relates to a method, composition and kit for radiolabeling proteins, including antibodies, with radioisotopes of technetium and rhenium such as tech- netium-99m.
2. Description of the Related Art Including Information Disclosed under 37 C.F.R. Sections 1.97-1.99 (Background Art)
The use of radioisotopes to label proteins is well known. These compositions can be used in assays, can be administered to the human body to visualize or monitor functioning of various parts of the body or to determine the presence and location of particular antigens, antibodies, hormones and the like, and can be used in the treatment of various disease states. A variety of radioisotopes, including isotopes of iodine, technetium, indium, and rhenium have been used. It is also well known that protein molecules can be tagged or labeled with technetium-99m to form a diagnostic or imaging agent.
Technetium-99m has been utilized to radiolabel proteins, chelating agents, phosphonate bone scanning compositions and the like by a technique which utilizes sodium pertechnetate wherein the technetium initially is in the +7 state. Technetium-99m is generally available only as sodium pertechnetate. The pertechnetate must be contacted with a reducmg agent, such as stannous chloride, in order to reduce the technetium to the +3, +4 or +5 oxidation state in the presence of the protein, chelating agent or like substance which is to be radiolabeled. The technetium must be maintained in this reduced state in order to maintain the chemical bond between the technetium molecule and the substrate being radiolabeled. It is also necessary that the technetium be firmly bound to the protein such that the reduced technetium is not transferred to other molecules or other proteins present in the assay, patient's blood or other media in which the radiolabeled substance will be utilized.
Several different methods have been utilized to radio-label proteins, particularly monoclonal antibodies, with technetium-99m. The methods involve two general approaches. One approach is indirect in which a bifunctional chelating agent is attached to the protein via one functional group and the technetium-99m is attached via the other functional, or chelating group. This method was introduced by Krejcarek. G. E. and Tucker. K. L. ("Covalent Attachment of Chelating Groups to Macromolecules," Biochemical and Biophysical Research Communications Vol. 77, pp. 581-585, 1977) and has been widely employed in many variations using a wide variety of bifunctional chelating agents such as described in the review of Wensel and Meares (Wensel, T. G., and Meares, C.F., "`Bifunctional` Chelating Agents for Binding Metal Ions to Proteins," Radioimmunoimaging and Radioimmunotherapy, S. W. Burchiel and B. A. Rhodes, eds., Elsevier Publishing Co., New York, pp. 185-196, 1983). Other methods are disclosed by Hnatowich, D. J., U.S. Pat. Nos. 4,668,503 and 4,479,930, by Haber, E., and Khaw, B. A., U.S. Pat. No. 4,421,735, by Fritzberg, A. R., and Kasina, S., U.S. Pat. No. 4,670,545 and by Baidoo, K. E., et al, ".sup.99m Tc Labeling of Proteins: Irtitial Evaluation of Novel Diaminedithiol Bifunctional Chelating Agent," Cancer Res (Supp), Vol. 50, pp. 799s-803s, 1990. The bifunctional chelate methods all present significant limitations, including the complexity of the radiolabeling procedure, the time required to accomplish radiolabeling, and the introduction and presence of substances which may affect the protein.
The other general approach is direct labeling. Although several direct methods have been reported, the fast direct method capable of providing a sufficiently strong bond between the protein and the technetium-99m for in vivo applications was the direct or pretinning method described in U.S. Pat. No. 4,424,200, entitled Method for Radiolabeling Proteins with Technetium-99m, to Crockford, D. R., and Rhodes, B. A. In this method, a single reduction solution, consisting of stannous [Sn (II)] chloride with tartrate and phthalate salts, is used. This solution serves to (1) reduce the protein, thereby exposing reactive sulfide groups, (2) protect the reactive sulfide groups of the reduced protein to prevent reformation of disulfide bonds, (3) reduce sodium pertechnetate, and (4) complex the reduced Tc-99m and transfer it to the sulfide binding sites of the reduced protein. With this method, many proteins can be successfully radiolabeled with technetium-99m. Several investigators have reported on the use of this method (Rhodes, B. A., et al, "Technetium-99m Labeling of Murine Monoclonal Antibody Fragments," Journal of Nuclear Medicine, Vol. 27, pp. 685-693, 1986: Some, P., et al, "Radioimmunoimaging of Experimental Thrombi in Dogs Using Technetium-99m-Labeled Monoclonal Antibody Fragments Reactive with Human Platelets," Journal of Nuclear Medicine, Vol. 27, No. 8, pp. 1315-1320, 1986).
Other early direct labeling methods were reported, but did not yield a stable Tc-protein bond. The reason for the instability of the Tc-protein bond in prior methods (Stern, H. S., et al., Radioactive Pharmaceuticals, U.S. Atomic Energy Commission (1966), Textbook, Chapter 19, pp. 359-375; Lin, M. S., et al., "Use of Fe (II) or Sn (II) Alone for Technetium Labeling of Albumin," Journal of Nuclear Medicine, Vol. 12, No. 5, pps. 204-211, 1970; Eckelman, W. C., et al., .sup.99m Tc-Human Serum Albumin," Journal of Nuclear Medicine, Vol. 12, No. 11, pp. 707-710, 1971; Wong, D. W., et al., "A Rapid Chemical Method of Labeling Human Plasma Proteins with .sup.99m Tc-Pertechnetate at pH 7.4," International Journal of Applied Radiation and Isotopes, Vol. 29, pp. 251-253, 1978; Colombetti, L. G., et al., "A Rapid Method for Labeling IgG with 99m-Tc," Journal of Nuclear Medicine, Vol. 20, p. 652, 1979; Rhodes, B. A., U.S. Pat. No. 4,305,922, Labeling Proteins with 99m-Tc by Ligand Exchange, was that the protein had not been reduced to provide reactive sulfide groups which are necessary for the formation of strong bonds between the protein and the reduced radionuclide.
Subsequently, a number of methods have been reported which employ variations on the method of U.S. Pat. No. 4,424,200. These variations generally involve one or more of the following: (1) Disulfide reducing agents other than Sn (II), such as 2-mercaptoethanol, dithiothreitol and their analogs, are used to reduce the protein; (2) reactive sulfide group protecting agents other than Sn (II), such as Zn (II), are employed; (3) pertechnetate reducing agents other than Sn (II), such as dithiothreitol, are used; and (4) complexing agents other than tartrate, such as phosphonates, are used to bind the reduced technetium and transfer it to the sulfide groups of the protein.
These methods have generally not resulted in any improvement over the method of U.S. Pat. No. 4,424,200. None of the methods disclosed yield results comparable to those achieved with the method, composition and kit disclosed herein.
Schwarz, A., and Steinstruaber, A., "A Novel Approach to Tc-99m-Labeled Monoclonal Antibodies," Journal of Nuclear Medicine, Vol. 28, p. 721, 1987, and Bremer, K. H., et al., European Patent Office Application No. 0 271 806 A2 filed Dec. 8, 1987), reduce the disulfide groups of the protein with monothiols, such as 2-mercaptoethanol or 2-mercaptoethylamine. Sn (II) is used to reduce the pertechnetate and the reduced technetium is complexed with phosphonates or pyrophosphates. This method requires two or more vials and multiple steps to achieve radiolabeling. The phosphonates used can give rise to radiocolloid impurities in the final product. In addition, the chemicals used to reduce the disulfide groups, such as 2-mercaptoethanol, are potentially toxic.
Reno, J. W., et al., U.S. Pat. No. 4,877,686 Radionuclide Antibody Coupling and European Patent Office Application No. 0 237 150 (filed Jan. 19, 1987), used dithiothreitol (DTT) to reduce the disulfide groups of the protein, then protect the reactive sulfides with Zn (II) or other sulfhydryl group derivatizing reagents. They use tartrate salts to complex and transfer the reduced radionuclide. This method uses potentially toxic chemicals, such as dithiothreitol, to reduce the antibody. It also requires multiple steps to radiolabel the protein.
Pak, K. Y., et al, "A Rapid and Efficient Method for Labeling IgG Antibodies with Tc-99m and Comparison to Tc-99m Fab' Antibody Fragments," Journal of Nuclear Medicine, vol. 30, p. 793, 1989, and Patent Cooperation Treaty International Patent Application No. WO 88/07382 (filed Apr. 1, 1988), used dithiothreitol to reduce the antibodies. Tartrate or glucoheptonate salts and their analogs are added to complex and transfer the reduced radionuclides. This method also uses potentially toxic chemicals, and requires multiple steps to radiolabel.
Shochat, D., et al., European Patent Office Application No. 0 336 678 (filed Apr. 3, 1989) use conventional disulfide reducing agents, such as cysteine, dithiothreitol, 2-mercaptoethanol, dithionite or the like. They claim that the pretinning method of U.S. Pat. No. 4,424,200 does not work well, indicating that some of the radiometal is bound to sites which are comparatively labile in the presence of blood or other bodily fluids or tissues. They give a single example in the application, preparation of Tc-99m-anti-CEA-Fab', which example appears to be exactly that of the '200 patent. The use of Sn (II) to reduce sodium pertechnetate is well known in the prior art, and is disclosed in the '200 patent and other references.
The advantage of the Sn (II) method of reducing disulfides of proteins over other methods is that the Sn (II) both reduces the bond and complexes with the sulfide formed by the reduction to protect the sulfide from reverting to unreactive disulfide. When organic reducing agents such as DTT are used to reduce the disulfide groups of the protein, the reducing agent must be removed before sulfide protecting groups are added, otherwise the protecting groups will react, usually by formation of a precipitate, with the reducing agent. If the reducing agent is first removed to avoid this reaction between it and the sulfide protecting agent, then the reduced protein is left for a period of time in which the reactive sulfide groups can reform unreactive disulfide bonds. The Sn (II) reduction method, described in this invention, is new in that it permits simultaneous reduction and complexing of disulfides.
Other methods of direct labeling have also been reported which differ chemically from the four-step process described above. Paik, C. H. et al., U.S. Pat. No. 4,652,440, Method of Stably Radiolabeling Antibodies with Technetium and Rhenium, label proteins by Sn (II) reduction in the presence of a strong chelating agent, DTPA, which competes for the reduced radionuclide. Only strongly bonded Tc-99m labeling of the protein occurs probably by binding to native free sulfhydryl groups of the protein. However, considerable amounts of Tc-99m-DTPA are also formed and must be removed before the labeled protein can be used. This method lacks the first step of reducing the disulfide bonds needed to achieve high yields of strongly bonded radionuclide.
Sundrehagen, E., Patent Cooperation Treaty International Patent Application No. WO 85/03231 (filed Jan. 18, 1985), used gentisic acid to stabilize the low concentrations of Sn (II) used to reduce the pertechnetate. This method is useful in minimizing radiochemical impurities such as radiocolloids and oxidized radionuclide. This method lacks the first step of reducing the disulfide bonds needed to achieve high yields of strongly bonded radionuclide.
Lees, R. S., U.S. Pat. No. 4,647,445, Radiolabelled Lipoproteins and Method of Making Same uses dithionite at pH 8 to 9 to reduce both pertechnetate and lipoproteins simultaneously. This method requires that the labeled product be purified by column chromatography to remove radionuclidic impurities prior to use. Breedveld, F. C., et al., "Imaging of Inflammatory Arthritis with Technetittm-99m-Labeled IgG," Journal of Nuclear Medicine, Vol. 30, No. 12, pp. 2017-2021, 1989, first reduce pertechnetate with hydrochloric acid and then extract, transfer, and reduce the radionuclide to dryness. The protein is added to the vessel containing the dry, reduced radionuclide. Labeling is achieved during a 60 minute incubation at 40.degree. C. This method requires extensive preprocessing of the radionuclide and is thus not readily applied to an instant kit process for labeling and formulating a drug.
McKenzie, I., et al., "Coupling of the .sup.99m Technetium-Nitrido Group to Monoclonal Antibody and Use of the Complexes for the Detection of Tumors in Mice," Journal of the National Cancer Institute, Vol. 77, pp. 431-439, 1986, and Patent Cooperation Treaty Patent Application No. WO 87/04164 (filed Jan. 6, 1987), reduce antibodies to provide free sulfhydryl group binding sites or introduce free sulfhydryl groups binding sites and label the sites with .sup.99m TcN(Cl).sub.4 --. The product requires purification by gel chromatography to remove radiochemical impurities prior to use.
All previous methods are limited because they fail to provide a one-step labeling kit and method which yields, within 15 minutes, an injectable product free of significant radiochemical impurities and a product in which the radionuclide is quantitatively and strongly bonded to the protein without altering immunoreactivity when the protein being labeled is an antibody. In addition, many previous methods employ potentially toxic chemicals. Although the pretinning method of Crockford and Rhodes was able to provide a rapid, one-step labeling process yielding approximately 85% of the radionuclide strongly bonded to the protein, the radiochemical purity has not been high enough for clinical application of all monoclonal antibodies, and some products require final purification prior to patient administration. In the present invention, both the optimum conditions for reducing the protein and the optimum conditions for reducing the radionuclide can be achieved while retaining the convenience of one-step labeling. This is achieved by adding steps to the manufacturing process. The protein is reduced with Sn (II) as in the original pretinning method described by Crockford and Rhodes. After reduction is completed, the reduced protein is put through a purification or complexing step to remove excess reducing agent and reaction by-products such as stannic chloride or other Sn (IV) agents. Alternately, an organic reducing agent is used to reduce the protein, the organic reducing agent and reaction products then removed, and Sn (II) is added to form a Sn (II) and sulfur containing protein complex. A Sn (II) radionuclide reducing agent is added to the reduced protein solution in concentrations and with complexing agents which are optimum for subsequent radiolabeling. The reduced protein together with the radionuclide reducing solution are aliquoted, frozen and optionally lyophilized for storage until needed for radiolabeling.
Another alternative is to reduce the antibody using the pretinning method of Crockford and Rhodes disclosed in the '200 patent at optimal concentration of reagents for reducing the protein and forming the Sn (II) and sulfur containing protein complex, and then diluting this solution with reagents to achieve conditions which are optimal for reducing the radionuclide and causing it to transfer to the protein.